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Article for the Removal of from Mine by Psychrotolerant and Mesophilic Microbes at Markedly Continental Climate Temperatures

Kuanysh N. Seitkamal 1,2,* , Nariman K. Zhappar 1,2, Valentin M. Shaikhutdinov 1, Aigerim K. Shibayeva 1,*, Sadia Ilyas 3, Ilya V. Korolkov 2,4 and Hyunjung Kim 3 1 LLP “Scientific-Analytical Center “Biomedpreparat”, Stepnogorsk, 3 microdistrict-9, Stepnogorsk, Akmola Region 021500, Kazakhstan; [email protected] (N.K.Z.); [email protected] (V.M.S.) 2 L.N. Gumilyov Eurasian National University, Satpaev str. 5, Nur-Sultan 010008, Kazakhstan; [email protected] 3 Department of Mineral Resources and Engineering, Jeonbuk National University, 567, Baekje-daero, Deokjin-gu, Jeonju, Jeonbuk 54896, Korea; [email protected] (S.I.); [email protected] (H.K.) 4 The Institute of Nuclear Physics, Ibragimov str., 1, Almaty 050032, Kazakhstan * Correspondence: [email protected] (K.N.S.); [email protected] (A.K.S.)

 Received: 25 September 2020; Accepted: 29 October 2020; Published: 31 October 2020 

Abstract: This study investigated the biological removal of heavy from mine tailings in Kazakhstan using acidophilic strains ferrivorans 535 and Acidithiobacillus ferrooxidans 377. The experiments were conducted in shake flasks at pH 1.6, various temperatures (28 ◦C, 18 ◦C, and 8 ◦C), and 10% solid concentration (w/v). The results of inductively coupled plasma optical emission spectroscopy and X-ray diffraction analyses showed that arsenic was particularly efficiently removed at 28 ◦C. At this temperature, A. ferrooxidans 377 was more efficient at removal than the other strain. Meanwhile, A. ferrivorans 535 was more efficient than A. ferrooxidans 377 at 8 ◦C. One of the more significant findings to emerge from this study is that arsenic can be removed at a low temperature and high solid concentration. The results of this study support the idea that can be used for removing arsenic via a combination of biooxidation and chemical methods.

Keywords: Acidithiobacillus ferrivorans; Acidithiobacillus ferrooxidans; arsenic removal; bioleaching; mine tailings

1. Introduction The control of hazardous waste, such as arsenic, is a major issue globally. Arsenic (As) is a hazardous waste product generated from the processing of various , such as , , , , and [1]. It is a ubiquitous element found at trace levels in all living matter, soil, rocks, natural , and the atmosphere. An increased level of arsenic can be mobilized through natural events such as volcanic emissions, especially the associated weathering products and ash, as well as anthropogenic activities. Humans have had significantly more influence on arsenic levels than natural events, through nonferrous and smelting, fossil fuel processing and combustion, wood preserving, pesticide production and application, and the disposal and of municipal and industrial waste [2]. This has resulted in the pollution of soil and [3]. The majority of arsenic compounds are tasteless, odorless, and easily dissolve in groundwater, which presents a risk to health. Arsenic, as both inorganic and organometallic species, naturally occurs in the environment essentially in four oxidation states ( III, 0, +III, and +V). Arsenite (As(III)) is about − 60 times more toxic than arsenate (As(V)). The exposure of individuals to arsenic mostly occurs

Minerals 2020, 10, 972; doi:10.3390/min10110972 www.mdpi.com/journal/minerals Minerals 2020, 10, 972 2 of 13 through drinking arsenic-contaminated [4]. According to the World Health Organization, the consumption of water and food containing more than 0.01 mg/L inorganic arsenic is harmful to the body, whereas consuming that with a level exceeding 60 mg/L can be fatal [5]. It has been reported that, in India and Bangladesh, 60–100 million people are at risk of arsenic-related diseases from drinking arsenic-contaminated water [6]. This is a particular issue in Bangladesh, where thousands of people are dying of arsenicosis [7]. Many cases of arsenic poisoning have been reported from around the world, such as in the USA, Canada, Poland, Taiwan, China, Bangladesh, Chile, Vietnam, Japan, India, Mexico, and Argentina [8–10]. Kazakhstan is one of the largest producers of hazardous waste in the world. It has accumulated over 22 billion tons of waste, of which more than 16 billion tons is mining and processing waste, and about 6 billion tons is hazardous waste [11]. Mining and processing complexes in the Karaganda (29.4%), East Kazakhstan (25.7%), Kostanay (17.0%), and Pavlodar (14.6%) regions have produced the largest proportions of the waste in the country. These mine tailings are deposited in tailings dumps and exposed to the environment at low temperatures of about 0 ◦C for five or more months each year. The extraction of valuable minerals from the earth and mineral processing operations produce large amounts of waste, which are deposited as waste dumps or tailings [12]. In mine tailings, arsenic has been shown to occur in various forms, such as arsenopyrite (FeAsS), arsenian Fe(As,S)2, and arsenates, and is known to associate with oxyhydroxides. Since arsenic has been shown to be related to gold deposits, gold mining may contribute to arsenic pollution. Indeed, gold mining activities were acknowledged to be the key source of arsenic contamination in many regions [13]. Thus, to reduce the health risks arising from As, it is necessary to develop strategies that could reduce its . Since As (V) is less soluble and is more effectively removed by physicochemical methods, it is important to oxidize As(III) to As(V) to achieve the satisfactory results of As removal [14]. Based on the analysis of data in the literature, using presently available technologies, especially physicochemical ones such as oxidation, , exchange, precipitation–coagulation, membrane filtration, permeable reactive methods, and biological techniques like and biological treatment with living microbes/bio-filtration, arsenic can be removed from contaminated water. However, all of these processes require an oxidation step to transform soluble As(III) to less soluble As(V), followed by the latter’s separation. Since oxidation via a reaction with under atmospheric conditions takes a long time, this step is usually performed using chemical oxidants such as ozone, hydrogen peroxide, and chlorine [15]. That explains why bioleaching, using various microorganisms (acidophilic or fungi), can be an environmentally friendly and economical method providing an alternative to traditional methods. Biological has been extensively studied over the past few years and is one of the used for the treatment of heavy metals (e.g., Cu, Co, Ni, Zn, and U) contained in sewage sludge, sediments, and contaminated soil [16]. Its use has been based on the ability of microorganisms to convert solid compounds into soluble elements that will be recovered. Recent studies have shown that high concentrations of arsenic can be leached from acid-contaminated soils by acidophilic iron- and -oxidizing bacteria. This has many advantages, such as low energy consumption, no emission of inorganic gaseous pollutants, increased leaching capacity, production of leaching agents in situ, and the formation of a microclimate around particles with high concentrations of leaching agents. The process also has certain disadvantages, such as longer reaction times, climate dependence, heavy metal toxicity, flotation, SX reagents for microbial activity, and the possibility of acid leakage [17]. The most familiar representatives of acidophilic bacteria used as bioleaching bacteria in leaching the metals in contaminated soils are bacteria from the genera Acidithiobacillus, Leptospirillum, Acidimicrobium, Sulfobacillus, and Sulfolobus [18]. Numerous studies have been conducted on bioleaching under mesophilic, moderately thermophilic, and extremely thermophilic conditions [19–21]. Acidithiobacillus is the most frequently used microorganism and studies on bioleaching mechanisms have focused mainly on Acidithiobacillus ferrooxidans (A. ferrooxidans)[22]. This species is a gram-negative acidophilic chemolithoautotroph, which requires atmospheric CO2 as a carbon source and obtains its energy for growth from the oxidation of ferrous (Fe2+) to ferric (Fe3+) Minerals 2020, 10, 972 3 of 13 iron [23–25]. A. ferrooxidans has been shown to be resistant to high concentrations of arsenic metalloid and is able to remove As in medium containing ferrous iron (Fe(II)) [22]. Hallberg et al. showed that, with several common phenotypic characteristics, Acidithiobacillus ferrivorans (A. ferrivorans) can grow at temperatures as low as 4 ◦C. They also found that A. ferrivorans can dominate in cold iron-rich environments with pH > 2.3. A. ferrivorans cells are gram negative, motile, and cannot form endospores, having an optimal temperature for growth of 27–32 ◦C[26]. It was found that ferrous iron can be more efficiently oxidized by the psychrotolerant mesophile A. ferrivorans in the bioleaching process of sulfide minerals at 4 ◦C than by the mesophilic A. ferrooxidans [27]. These oxidizing properties of bacteria are useful in arsenic removal processes. In the reaction with dissolved Fe(III) at low pH, As(III) can be oxidized to As(V) according to Reactions (Equations (1) and (2)) [28].

3+ + 2+ H AsO + H O + 2Fe H AsO− + 3H + 2Fe (1) 3 3 2 → 2 4 2H AsO + Fe (SO ) 2FeAsO + 3H SO (2) 3 4 2 4 3 → 4 2 4 In previous studies on bioleaching, tests using A. ferrivorans were conducted [29,30]. However, in cold environments such as that in countries with a markedly continental climate, including Kazakhstan, low temperature becomes a limiting factor, reducing leaching speed and potentially making the process uneconomical. Nonetheless, the oxidation of sulfide minerals at a low temperature (<20 ◦C) has barely been studied. Bioleaching at a low temperature was reported in only a few papers [31–35]. However, bioleaching of arsenic-containing mine tailings at a low temperature (<10 ◦C), especially bioleaching by A. ferrivorans, has not been studied. Therefore, in the present work, A. ferrivorans strain 535 and A. ferrooxidans strain 377 were used for bioleaching in mine tailings containing As at temperatures of 8 ◦C, 18 ◦C, and 28 ◦C, which are common in countries with a markedly continental climate. The effects of ferric iron on the bioleaching kinetics of arsenic in a from waste were studied. During the bioleaching process, we investigated the relationships between the parameters of pH, oxidation–reduction potential (ORP), and As concentration. The study provides new information about the mechanisms of biological leaching of arsenic at low temperature.

2. Materials and Methods

2.1. Microorganisms and Samples

In this study, mine tailings were collected for testing from Bestobe Mine (52◦3601800 N, 73◦1304800 E), Akmola, Kazakhstan, which were kindly provided by Kazakhaltyn Mining-Metallurgical Concern JSC (Stepnogorsk, Akmola region, Kazakhstan). The 377 strain of A. ferrooxidans and the 535 strain of A. ferrivorans that were previously isolated from gold deposits at our laboratory were used in this study. These isolates were registered with the official potent deposit service at Republican State Enterprise “Republican collection of microorganisms” of the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (http://rcm.kz/) in Nur-Sultan, Kazakhstan. Microorganisms were incubated in 100 mL of Medium 9K-Fe [36] in 500 mL flasks under aerobic conditions at 28 ◦C and 200 rpm. The medium was adjusted to pH 1.8 at room temperature using 10 N H2SO4 and autoclaved at 121 ◦C for 20 min.

2.2. Analytical Analysis The mine tailings before and after bioleaching were analyzed mineralogically by X-ray diffraction (XRD) on a D8 ADVANCE (Bruker AXS GmbH, Billerica, MA, USA). Chemical phase analysis of the mine tailings for the arsenic and iron forms was carried out in accordance with the methods described by Filippova [37]. Minerallographic analyses of the mine tailings were conducted using the microscope OLYMPUS BX-51 Pol (Olympus Corporation, Shinjuku City, Tokyo, Japan) with a SIMAGIS 2P-2C video camera (SIAMS Ltd., Ekaterinburg, Russia) and SIAMS’ Mineral S-7 image analysis software (SIAMS Ltd.). The minerallographic analyses were carried out in the accredited laboratory (ISO 17025:2007) of Minerals 2020, 10, 972 4 of 13

Eastern Research Mining and Metallurgical Institute of Nonferrous Metals. The polished samples for XRD analyses were 70 microns. The volume of the samples were 0.2–0.5 cm3. It must also be stable and not decompose for at least 2 h. The method for preparing polished samples for microscopy was performed as described previously [38]. The mine tailings were also analyzed by inductively coupled plasma optical emission spectroscopy (ICP-OES, Thermo Scientific iCAP 7200 ICP Duo, Waltham, MA, USA). The chemical composition of the mine tailings was determined by adding 8 mL of 35% hydrochloric acid, 2 mL of 45% hydrogen fluoride, and 0.50 mL of 65% nitric acid to 0.25 g of the mine tailings. After that, the samples were heated at about 150 ◦C for 1 h, then kept at room temperature and diluted. Next, the suspension was introduced to ICP-OES for analysis of the target elements. During the tests, 1 mL samples of the bioleaching solution were periodically taken (every 2 days and then every 4 days from 6 days after the bioleaching tests) from each flask under sterile conditions. The collected samples were each filtered and centrifuged for analysis of arsenic, ferric, and ferrous ion concentrations. The concentration of arsenic was analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES, Thermo Scientific iCAP 7200 ICP Duo). The concentrations of ferric and ferrous in the solution were determined by spectrometry (BioMate 3S UV-Visible, Thermo Scientific, Waltham, MA, USA). The pH and redox potential of the leaching solutions were periodically analyzed using a saturated calomel electrode (Mettler Toledo Seven Multi S47-K, Mettler Toledo AG, Schwerzenbach, Switzerland), while redox potential (Eh) was measured in units of mV by the combination of a platinum ring indicator and S7 screw head. All chemical analyses of samples and solutions were performed by the Biomedpreparat Scientic Analytical Center (Stepnogorsk, Akmola region, Kazakhstan), which is an accredited laboratory (ISO 17025:2009).

2.3. Bioleaching Experiments The bioleaching experiments were conducted in 250 mL Erlenmeyer flasks containing 10% mine tailings (w/v) and 10% inoculum (v/v) in a shaking incubator at 160 rpm and temperatures of 8 1 C, ± ◦ 18 1 C, and 28 1 C. The initial pH of modified medium Fe-9K supplemented with filter-sterilized ± ◦ ± ◦ FeSO 7H O (22.1 g/L) was adjusted to 1.6 using 10 N H SO During the As leaching tests, the pH 4· 2 2 4. of each flask decreased to the initial level of 1.6. The pH of each flask was periodically controlled and adjusted to the initial pH to prevent the formation of jarosite (KFe3(SO4)2 (OH)6)[39]. The initial concentrations of the added inocula were determined by direct counting under a phase-contrast microscope (Standard 25, Carl Zeiss, Oberkochen, Germany) and by serial dilutions. Control tests without the microorganisms were also carried out under the same conditions. The final efficiency of As removal was calculated considering the mass balance of metal concentrations in solid and liquid phases. The particle size distribution of mine tailings used for bioleaching tests was meshed until 142 µm. During the bioleaching process, a weight difference method was used by replacing lost water with distilled water. All bioleaching processes were carried out for 32 days.

3. Results and Discussion

3.1. Ore Sample Characterization The chemical analysis on the sample showed that the total content of As was 0.045, that of S was 0.15, that of Fe was 3.1, and that of Zn was <0.05 (Table1). XRD showed that the sample mainly contained quartz (SiO ), scorodite (FeAsO 2H O), albite, ordered (NaAlSi O ), muscovite-2M1 2 4· 2 3 8 glicolated [KAl2(Si,Al)4O10(OH)2], ferroan dolomite [Ca(Mg,Fe)(CO3)2], and ferroan clinoclore [Mg5Al(AlSi3O10)(OH)8] (Figure1. As it was di fficult to determine arsenic-containing minerals using XRD, minerallographic analysis and phase chemical analysis were performed. The results obtained from the minerallographic analysis are presented in Figure2. Arsenopyrite was found in the free forms with a particle size of 0.05 mm and in the non-ore forms with a wide size range from 0.005 to 0.025 mm. and pyrite were found in the nonmetallic mass of the rock. According to the phase chemical analysis (Table2), it was determined that the arsenic is for the most part represented by Minerals 2020, 10, 972 5 of 13 arsenopyrite and scorodite minerals with a mass fraction (%) of 0.022. The mass fraction (%) of arsenic as oxide,Minerals elemental, 2020, 10, 972 and sulfide forms was not more than 0.005. The total mass fraction of Fe5 was of 13 3.1%. Minerals 2020, 10, 972 5 of 13 TableTable 1. The1. The selected selected elemental elementalcontent content of mine tailings tailings determined determined by by ICP ICP-OES-OES analysis analysis.. Table 1. The selected elemental content of mine tailings determined by ICP-OES analysis. ElementsElements As As Fe Fe S SZnZn Composition (%) 0.045 3.10 0.15 <0.05 CompositionElements (%) 0.045As Fe3.10 S 0.15 Zn< 0.05 Composition (%) 0.045 3.10 0.15 <0.05

Figure 1. XRD pattern of mine tailings before bioleaching. Figure 1. Figure 1.XRD XRD patternpattern of mine tailings tailings before before bioleaching. bioleaching.

(a) (b) (a) (b)

(c)

(c) FigureFigure 2. Optical 2. Optical microscopy microscopy (x200) (x200) images images of theof the minerals minerals before before bioleaching: bioleaching: Polished Polished section section of of mine tailings,mineFigure (atailings, )2. Single Optical ( graina) microscopySingle of arsenopyrite;grain (x200) of arsenopyrite; images (b) Arsenopyrite of the(b) mineralsArsenopyrite in non-ore before in bioleaching: forms;non-ore ( cforms;) Pyrite Polished (c (light) Pyrite section yellow) (light of in a non-oreyellow)mine fragment;tailings, in a non (a hematite-)ore Single fragment; grain (gray). hematiteof arsenopyrite; (gray). (b) Arsenopyrite in non-ore forms; (c) Pyrite (light yellow) in a non-ore fragment; hematite (gray).

Minerals 2020, 10, 972 6 of 13

3.2. Bioleaching Experiments To examine the effects of microorganisms at a low temperature as well as other temperatures, arsenic bioleaching experiments were conducted at initial pH of 1.6 and pulp density of 10% with temperatures of 28 ◦C, 18 ◦C, and 8 ◦C by A. ferrooxidans, A. ferrivorans, and uninoculated control. Since the A. ferrooxidans and A. ferrivorans bacteria are acidophilic microorganisms active at an acidic pH, the pH level during the bioleaching process with different bacterial cultures did not exceed 1.8. Changes in the pH of all strains were reasonably similar. In the flasks with A. ferrooxidans 377 and A. ferrivorans 535 at 28 ◦C and 8 ◦C, respectively, the pH went down to 1.6. Meanwhile, the pH did not drop notably in the flasks without bacterial inoculation (data not shown). The As removal rates, reduction potential, and iron concentrates at 28 ◦C are shown in Figure3. Since Bestobe Mine tailings contain arsenic in the form of arsenopyrite (FeAsS), arsenic removal kinetics in terms of the biological leaching of FeAsS was analyzed. In terms of the biological oxidation mechanisms, the bacteria oxidize ferrous iron to ferric iron and the As(III) can be oxidized to As(V) by ferric iron, according to Reactions (Equations (1), (3) and (4)) [40]:

4Fe2+ + 4H+ + O 4Fe3+ + 2H O (3) 2 → 2 FeAsS + 5Fe3+ + 3H O 2H AsO + 6Fe2+ + S0 + 3H+ (4) 2 → 3 3 In a study conducted by Zhang et al., it was shown that, during bioleaching tests, the oxidation–reduction potential (ORP) and pH values are the two most important factors, wherein at low pH and high ORP Fe(III) disturbs the surface of arsenopyrite by producing Fe(II) (Equation (4)). Thereafter, the accumulation of Fe(III), arsenate, and some sulfates forms several Fe(III)-containing precipitates (jarosites) [41]. The ferrous iron to ferric iron oxidation, which is described in the first , is assisted by bacteria. In the second reaction, the arsenopyrite is oxidized by free ferric ions with the dissolving arsenopyrite to form arsenic acid. The As bioleaching rate was remarkably increased in the flasks with microorganisms for 14 days, but the As concentration in the solution subsequently increased slowly, changing by 2–3%. Meanwhile, in the control, the As removal efficiency of the reaction increased to about 20% in 26 days, but no subsequent As leaching was observed in that reaction. According to these results, the effect of microorganisms might have been inhibited in As leaching. In addition, the strain A. ferrivorans 535 achieved the highest redox potential after 10 days, while A. ferrooxidans 377 reached it in 4 days. Strains A. ferrooxidans 377 showed the highest activity, the active oxidation of iron in the medium was observed for 4 days, and the ORP values were above 550 mV. Although the strains A. ferrivorans 535 and A. ferrooxidans 377 oxidized 99% of Fe2+ to Fe3+ after 10 days, the bioleaching was continued till the end the experiments. After 18 days of bioleaching, the levels of Fe3+ and ORP began to decrease. Meanwhile, Fe2+ was increased, which indicates a decrease in activity due to an increase in the content of arsenic in the leaching solution above 31 mg/L (61%). The arsenic dissolution curves show that the efficiency of leaching As was directly related to the oxidation of Fe2+ in solution. Throughout the experiment, in all flasks with microorganisms, the ORP was kept above 510 mV while the redox potential of the control flasks varied between 393 and 444 mV. The bioleaching efficiency of arsenic was 66–68%. A long period of adaptation of strains to the mine tailings in all flasks at 18 ◦C was observed (Figure4). Strains A. ferrivorans 535 showed better results regarding iron oxidation and arsenic recovery than strains A. ferrooxidans 377. In the flask with the strains A. ferrivorans 535, the complete oxidation of Fe2+ was achieved in 14 days. Along with the oxidation of the Fe2+, an increase in redox potential of the medium occurred, where the ORP value rose to 640 mV. In addition, strains A. ferrivorans 535 removed 64% of arsenic in 32 days. Meanwhile, strains A. ferrooxidans 377 was less active, as the complete oxidation of iron occurred in 19 days. Minerals 2020, 10, 972 7 of 13 MineralsMinerals 20202020,, 1010,, 972 972 7 7of of 13 13

80 0 (b) A.ferrivorans 535 (a) 28 С80 600 A. ferrooxidans 377 A.ferrivorans 535 0 (b) 6 Control (a) 28 С 600 A. ferrooxidans 377 6 Control 60 550 60 550 4 , g/l 500 43+ 40 As, % , g/l

Fe 500

3+ 40 Redox, mV Redox, As, %

Fe

2 mV Redox, 450 20 2 450 20 400 0 0 400 0 4 8 12 16 20 24 28 32 0 5 10 15 20 25 30 0 Time, days 0 0 4 8 12 16 20 24 28 32 0 5 10 Time,15 days20 25 30 Time, days Time, days Figure 3. Ferric (Fe3+) concentration (a), solution redox potential, and As removal efficiency (b) during 3+ FigureFigurethe bioleaching 3. 3. FerricFerric (Fe (Fe experiments3+) concentration) concentration with ( adifferent (),a), solution solution strains redox redox at potential, potential,28 °C. and and As As removal removal efficiency efficiency ( (bb)) during during thethe bioleaching bioleaching experiments experiments with with different different strains at 28 ◦°C.C.

7 80 0 700 7 A.ferrivorans 535 18 80С A. ferrooxidans 377 0 700 6 A.ferrivorans Control 535 18 С A. ferrooxidans 377 6 Control 60 5 60 600 5 600 4 , g/l

43+ 40 As, % , g/l

Fe 3 3+ (a) 40 500 (b) As, % Redox, mV Redox,

Fe 3 (a) 500 (b)

2 mV Redox, 20 2 20 1 400 1 400 0 0 -4 0 4 8 12 16 20 24 28 32 36 0 5 10 15 20 25 30 0 Time, days 0 -4 0 4 8 12 16 20 24 28 32 36 0 5 10 15Time, days20 25 30 Time, days Time, days FigureFigure 4. 4.Ferric Ferric (Fe (Fe3+3+)) concentration concentration (a (),a) solution, solution redox redox potential, potential, and and As As removal removal effi efciencyficiency (b ()b during) during 3+ Figurethethe bioleaching bioleaching 4. Ferric (Fe experiments experiments) concentration with with di ( ffadifferent)erent, solution strains strains redox at 18at potential, 18◦C. °C. and As removal efficiency (b) during the bioleaching experiments with different strains at 18 °C. TheThe changes changes in in iron iron oxidation, oxidation, ORP, ORP, as as well well as as As As concentration concentration for for the the temperature temperature of of 8 ◦8C °C are are shownshownThe in changesin Figure Figure5 .in 5.A. iron A. ferrivorans ferrivorans oxidation,535 535 ORP, showed showed as well higher higher as As activity activityconcentration than thanA. A. ferrooxidansfor ferrooxidans the temperature377, 377, in in line ofline with8 °Cwith are the the showndidifferencesfferences in Figure observed observed 5. A. betweenferrivorans between the the535 two two showed strains strains higher by by other other activity researchersresearchers than A. [[26ferrooxidans26––2929]].. The 377,complete complete in line oxidation oxidation with the of differencesofiron iron by by A.A. observed ferrivorans between 535 occurred the two instrains 1818 days,days, by other andand theresearchersthe ORPORP valuevalue [26 rose– rose29]. above Theabove complete 620 620 mV. mV. oxidation The The arsenic arsenic of ironbioleachingbioleaching by A. ferrivorans within within the the535 32 32 daysoccurred days of of the thein experiment 18 experiment days, and was was the 61.8% 61.8%ORP (30.9 value(30.9 mg mrose/g/L).L Meanwhile,).above Meanwhile, 620 mV. in in the Thethe flask flaskarsenic with with bioleachingstrainstrain 377, 377, the within the complete complete the 32 oxidation days oxidation of the of ofexperiment iron iron was was achieved achievedwas 61.8% in in only (3 only0.9 22 m 22 days.g/ days.L). Meanwhile, in the flask with strainAlthough 377,Although the complete most most arsenic arsenic oxidation in in the the of mine ironmine tailingswas tailings achieved presented presented in only as as dissoluble22 dissoluble days. minerals minerals and and our our findings findings coincidecoincideAlthough with with most those those arsenic of of other other in researchersthe researchers mine tailings that that the presentedthe bioleaching bioleaching as dissoluble rates rates of of Asminerals As and and Fe andFe depend dependour findings on on the the coincidetemperature,temperature, with thethose the As As of removal removalother researchers effi efficiencyciency was thatwas more themore thanbioleaching than 50% 50% at atrates all all temperatures. temperatures.of As and Fe At Atdepend the the end end on of ofthe the the temperature,experiment,experiment, Asthe As removal Asremoval removal e ffiefciencyfi efficiencyciency reached reached was themore the highest highest than value50% value at (66.5%) all(66.5%) temperatures. by byA. A. ferrivorans ferrivorans At the535 535end at atof 28 28the◦ C.°C. experiment,AfterAfter 14 14 days days As at atremoval 28 28◦C, °C, As Asef leachingfi leachingciency didreached did not not change the change highest markedly. markedly. value The(66.5%) The same same by situation situationA. ferrivorans was was observed observed535 at 28 at at°C. the the Afterotherother 14 temperatures temperatures days at 28 °C, after after As 22 leaching 22 days. days. There didThere not are are change several several markedly. possible possible reasons Thereasons same for for situation the the low low As Aswas removal removal observed e ffiefficiency. ciency.at the otherFirst,First, temperatures the the toxic toxic eff effectect after of of arsenic 22 arsenic days. would Therewould increaseare increase several to to inhibitpossible inhibit bacterial reasonsbacterial growth.for growth. the low However, However, As removal several several efficiency. reports reports First,havehave the described described toxic effect that, that, of at arsenicat As As concentrations concentrations would increase of of 20to 20 ginhibit /g/LL or or higher,bacterial higher, bacterial bacterialgrowth. growth However,growth was was several inhibited inhibited reports [40 [40]]. . haveSecond,Second, described according according that, to toat Astudillo AstudilloAs concentrations et e al.t al. [42 [42]], at , ofat a higher20a higher g/L solidor solid higher, concentration, concentration, bacterial agrowth limitationa limitation was in inhibited in the the supply supply [40] of. of

Second,gaseousgaseous according nutrients nutrients suchto such Astudillo as as CO CO2 ore2 tor al. O O2[42]could2 could, at occura occurhigher due due solid to to the theconcentration, accumulation accumulation a oflimitation of soluble soluble ironin iron the and andsupply arsenic. arsenic. of gaseousThird,Third, the nutrientsthe formation formation such of jarositeofas jarosite CO2 occurredor occurredO2 could at a atoccur pH a pH of due 1.8 of or1.8to higher theor higher accumulation on the on mineralthe minera of soluble surface.l surface. iron The Theand formation formationarsenic. of Third,theof passivation the the passivation formation layer of bylayer jarosite jarosite by occurred (Equationjarosite at(Equation (5)) a pH could of 1.8 decrease(5) or) highercould the ondecrease leached the minera proportionthe lleached surface. [43 ].Theproportion According formation [43] to . ofthe Accordingthe arsenic passivation leaching to the arsenic inlayer the controlby leaching jarosite test, in Deng(Equation the control et al. pointed(5) test,) could Deng out decrease thatet al. the pointed relatively the leachedout high that arsenicproportionthe relatively extraction [43] high. Accordingarsenic extraction to the arsenic might leaching be due toin the the easy control test, Deng of arsenopyrite et al. pointed in acidic out that solutions the relatively and the high buffer arsenicproperties extraction of the might medium be due solution to the containingeasy solubility K2HPO of arsenopyrite4 [44]. in acidic solutions and the buffer properties of the medium solution containing K2HPO4 [44].

Minerals 2020, 10, 972 8 of 13 might be due to the easy solubility of arsenopyrite in acidic solutions and the buffer properties of the mediumMinerals solution 2020, 10, containing 972 K2HPO4 [44]. 8 of 13

+ 3+ 2 + K ++ 3Fe 3+ + 2SO2− − + 6H O KFe (SO ) (OH)+ + 6H (5) K + 3Fe + 2SO4 4 + 6H2O →2 KFe→ 3(SO43)2 (OH)4 26 + 6H 6 (5)

7 100 A.ferrivorans 535 80С A. ferrooxidans 377 6 Control 80 5

4 60 , g/l 3+ As, %

Fe 3 40 2 (a)

1 20

0 0 4 8 12 16 20 24 28 32 Time, days

700

600

500 Redox, mV Redox, (b)

400

0 4 8 12 16 20 24 28 32 Time, days FigureFigure 5. Ferric 5. Ferric (Fe3 (Fe+) concentration3+) concentration (a (),a), solution solution redox potential, potential, and and As As removal removal effi eciencyfficiency (b) during (b) during the bioleachingthe bioleaching experiments experiments with with di ffdifferenterent strains strains at at 8 8◦ °C.C.

TableTable 2. The 2. The results results of of the the phase phase chemicalchemical analysis analysis of ofAs Asand andFe in Fe the in mine the tailings mine tailingsbefore and before after and after bioleaching.bioleaching.

MassMass Fraction Fraction (%) (%) As Phases Before Leaching 8 °C 28 °C As Phases Before Leaching 8 C 28 C 377 535◦ Control 377 535 ◦Control as oxide <0.005 ND377 ** ND 535 ** ND Control ** ND ** 377 ND ** 535ND **Control as oxideas elemental <0.005<0.005 NDND ** ** ND ND ** ** ND ND ** **ND ND ** **ND ** ND ND ** ** ND ** as elementalassociated with Zn <0.005<0.005 NDND ** ** ND ND ** ** ND ND ** **ND ND ** **ND ** ND ND ** ** ND ** associated withas Zn <0.005<0.005 NDND ** ** ND ND ** ** 0.006 ND **ND ND ** **ND ** ND **0.007 ND ** asas difficult sulfide to decompose * <0.0050.022 ND0.022 ** 0.019 ND ** 0.023 0.006 0.015 ND **0.013ND **0.022 0.007 as difficult to decomposeTotal * 0.0220.045 0.0220.022 0.019 0.019 0.039 0.023 0.015 0.015 0.013 0.0130.034 0.022 TotalFe Phase 0.045 0.022 0.019 0.039 0.015 0.013 0.034 as oxide 3.1 2.11 2.03 1.59 1.71 1.86 1.4 Fe Phase as sulfide <0.005 0.48 0.48 0.73 2.41 2.55 0.09 as asdifficult oxide to decompose * 3.13.1 2.110.012 0.007 2.03 0.014 1.59 0.026 1.71 0.008 1.860.02 1.4 as sulfideTotal <0.0050.045 0.482.59 2.51 0.48 2.32 0.73 4.12 2.41 4.41 2.551.49 0.09 as difficult to decompose * 3.1 0.012 0.007 0.014 0.026 0.008 0.02 *- arsenopyrite, scorodite, jarosite (only in leach residue); **- not detected or <0.005. Total 0.045 2.59 2.51 2.32 4.12 4.41 1.49 Against *this arsenopyrite, background, scorodite, XRD jarosite analysis (only was in leachperformed residue); to **con notfi detectedrm the formation or <0.005. of jarosite on the mineral surface in mine tailings after bioleaching tests. It was shown that jarosite was not formed

Minerals 2020, 10, 972 9 of 13

Against this background, XRD analysis was performed to confirm the formation of jarosite on the mineralMinerals surface 2020, 10, 972 in mine tailings after bioleaching tests. It was shown that jarosite was not formed9 of 13 in low-temperature conditions (Figure6b). The jarosite intensity was observed at 28 ◦C temperatures in in low-temperature conditions (Figure 6b). The jarosite intensity was observed at 28 °C temperatures the flasks with all strains (Figure6a). From the experiments, we concluded that jarosite precipitation in the flasks with all strains (Figure 6a). From the experiments, we concluded that jarosite would not occur at a temperature of 8 C at the initial pH of 1.6; consequently, As leaching efficiency precipitation would not occur at a temperature◦ of 8 °C at the initial pH of 1.6; consequently, As was increased. leaching efficiency was increased.

(a)

(b)

Figure 6. XRD pattern of mine tailings after bioleaching in the flasks with strains A. ferrivorans 535 at Figure 6. XRD pattern of mine tailings after bioleaching in the flasks with strains A. ferrivorans 535 at 28 C(a) and 8 C(b). 28◦ °C (a) and ◦8 °C (b). In addition to the XRD analysis, arsenic and iron phase analysis was conducted with some residues In addition to the XRD analysis, arsenic and iron phase analysis was conducted with some after biological leaching tests. The results of the phase chemical analysis are depicted in Table2. In the residues after biological leaching tests. The results of the phase chemical analysis are depicted in TableTable2, data2. In ofthe the Table leach 2, d residueata of the can leach be comparedresidue can with be compared the data beforewith the leaching. data before What leaching can be. What clearly seencan in be the clearly table seen that in the the bioleaching table that the process bioleaching removed process a significant removed a part signi offi thecant arsenic part of asthe dissoluble arsenic andas didissolublefficult to and dissolve difficult minerals to dissolve at 28 minerals◦C. Meanwhile, at 28 °C. Meanwhile, at the low temperature at the low temperature of 8 ◦C, the of amount8 °C, ofthe arsenic amount as di offfi arseniccult to decomposeas difficult to hardly decompose changed. hardly All changed. of the results All of support the results the mechanismsupport the of bioleachingmechanism proposed of bioleaching in Figure proposed7. To obtainin Figure more 7. To precise obtain information more precise on information the stability on and the toxicitystability of theand bioleaching toxicity of residue, the bioleaching additional residue, studies, additional including studies, designing including a pilot designing scale, will a pilot be carried scale, will out. be carriedDespite out. the implications and significance of our research, there are some limitations in terms of the methods applied. One limitation is that we could not provide data on the growth phase of culture, or data from scanning microscopy. Another important limitation is that the leached arsenic needs to be removed by chemical methods such as sedimentation and filtration. To develop a comprehensive overview of the arsenic removal process, additional studies will be required on As(V) removal by chemical methods.

Minerals 2020, 10, 972 10 of 13 Minerals 2020, 10, 972 10 of 13

FigureFigure 7.7.The The mechanisms mechanisms of of bioleaching bioleaching of arsenopyriteof arsenopyrite from from the mine the tailingsmine tailings for the for removal the re ofmoval arsenic. of arsenic. 4. Conclusions InDespite this study, the implications bioleaching and for significance the removal of ofour arsenic research, using there biooxidation are some limitations by pure culturesin terms ofof A.the ferrooxidans methods applied.and A. ferrivoransOne limitationwas is investigated that we could using not mineprovide tailings data containingon the growth arsenic. phase The of culture, results or data from scanning electron microscopy. Another important limitation is that the leached arsenic of this investigation show that at initial pH of 1.6, at different temperatures (8 ◦C, 18 ◦C, and 28 ◦C), andneeds a solidto be concentration removed by ofchemical 10%, a decreasemethods in such the temperatureas sedimentation affected and the filtration. given microorganisms To develop a activitycomprehensive and the periodoverview of adaptationof the arsenic to theremoval mine process, tailings. additional After 32 days studies of cultivation, will be required at temperature on As(V) removal by chemical methods. of 28 ◦C the A. ferrooxidans 377 was showed the highest leaching efficiency of As (up to 68%) with Fe2+ iron oxidation of 4.7 g/L. In the bioleaching of mine tailings experiments at temperature of 8 C, 4. Conclusions ◦ the highest As leaching (61%) was observed in the flask with the A. ferrivorans with Fe2+ iron oxidation of 4.9In g/ L.this The study, As leaching bioleaching behaviors for the in allremoval the pure of culturesarsenic using according biooxidation to the experiment by pure cultures temperatures of A. showedferrooxidans that and a higher A. ferrivorans temperature was investigated to a tendency using mine to highertailings As containing leaching earsenic.fficiency. The The results study of contributesthis investigation to our understandingshow that at initial of the pH impact of 1.6, of at the different psychrotolerant temperatures andmesophilic (8 °C, 18 °C, microbes and 28 °C), in mine and tailingsa solid concentration during the removal of 10%, of a arsenicdecrease under in the low temperature temperature affected conditions, the giv anden microorganisms the influence of jarositeactivity formationand the period at low of and adaptation mesophilic to the temperatures. mine tailings. Overall, After this32 days study of strengthenscultivation, theat temperature idea that the of strains 28 °C oftheA. A. ferrivorans ferrooxidanscan 377 be morewas showed effective the than highest strains leaching of A. ferrooxidans efficiencyin of bioleaching As (up to 68%) of sulfide with mineralsFe2+ iron andoxidation tailings of at 4.7 low g/L. temperature. In the bioleaching Moreover, of mine the resultstailings reported experiments here at shed temperature new light of on 8 the °C, applicationthe highest ofAsA. leaching ferrivorans (61%)in lowwas temperatureobserved in bioleachingthe flask with of the arsenic-containing A. ferrivorans with mine Fe2+ tailings. iron oxidation Further of research 4.9 g/L. couldThe As also leaching be conducted behaviors to determine in all the pure the e ffculturesectiveness according of the use to ofthe a e consortiumxperiment oftemperatures strains in markedly showed continentalthat a higher climatic temperature conditions. leads to a tendency to higher As leaching efficiency. The study contributes to our understanding of the impact of the psychrotolerant and mesophilic microbes in mine tailings Authorduring Contributions:the removal ofConceptualization: arsenic under low K.N.S., temperature N.K.Z. and conditions, V.M.S.; Investigation: and the influence K.N.S. andof jarosite A.K.S.; Methodology: N.K.Z. and V.M.S.; Project administration: A.K.S.; Resources: K.N.S., N.K.Z., V.M.S. and A.K.S.; Supervision:formation at A.K.S.; low Validation:and mesophilic K.N.S., temperatures. N.K.Z. and V.M.S.; Overall, Visualization: this study I.V.K.; strengthens Writing—original the idea draft: that K.N.S.; the Writing—reviewstrains of A. ferrivorans and editing: can N.K.Z., be more V.M.S., effective S.I., A.K.S., than I.V.K.strains and of H.K. A. ferrooxidans All authors havein bioleach read anding agreed of sulfide to the publishedminerals versionand tailings of the at manuscript. low temperature. Moreover, the results reported here shed new light on the Funding:applicationThis of research A. ferrivorans was funded in bylow The temperature Science Committee bioleaching of the Ministryof arsenic of Education-containing and mine Science tailings. of the RepublicFurther ofresearch Kazakhstan could (grant also no.be conducted AP05136008). to determine the effectiveness of the use of a consortium of Acknowledgments:strains in markedlyFinancial continental supports climatic from Theconditions. Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan is gratefully acknowledged. We also gratefully acknowledge the donation of the ore sample from the Kazakhaltyn Mining-Metallurgical Concern JSC. Author Contributions: Conceptualization: K.N.S., N.K.Z. and V.M.S.; Investigation: K.N.S. and A.K.S.; ConflictsMethodology: of Interest: N.K.Z.The and authors V.M.S; declareProject noadm conflictinistration: of interest. A.K.S.; Resources: K.N.S., N.K.Z., V.M.S. and A.K.S.; Supervision: A.K.S.; Validation: K.N.S., N.K.Z. and V.M.S.; Visualization: I.V.K.; Writing—original draft: K.N.S.; Writing—review and editing: N.K.Z., V.M.S, S.I., A.K.S., I.V.K. and H.K. All authors have read and agreed to the published version of the manuscript.

Minerals 2020, 10, 972 11 of 13

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